Summary: Project 1 Ewing Sarcoma cells harbor rearrangements of the EWS gene with ETS domain transcription factors and are driven by the resulting EWS-fusion proteins. Therapeutic targeting of the fusion protein is the most rational potential therapeutic approach, but no small molecules are available to inactivate EWS-fusion protein function. However, recent developments toward the understanding of the mechanism of action of EWS-fusion proteins, and in the development of small molecules that inactivate difficult drug targets make this an opportune time to undertake a project focused on targeted inactivation of fusion oncoproteins. The primary goals of this project are to delineate the cellular effects that result from acute degradation of the EWS-fusion proteins, and to develop novel small molecules that directly target and ultimately degrade EWS-fusion proteins. In aim one, we will develop tools and use approaches that have been successfully used for the study of leukemia to interrogate the biology of EWS-fusion degradation. These studies will set the stage for experiments performed in Aims two and three that will focus on how to inhibit and/or degrade EWS-fusion proteins with newly developed small molecules. Aim 2 will develop cell lines that allow direct assessment of EWS-FLI1 fusion protein stability, and these cell lines will be used to identify the cellular processes in Ewing Sarcoma cells that regulate EWS-FLI1 protein levels. Understanding how Ewing Sarcoma cells naturally maintain protein levels will potentially allow targeted therapeutic development of molecules that destabilize this fusion oncoprotein. Furthermore, such cell lines will allow for high-throughput assessment and identification of molecules that lead to loss of the EWS-FLI1 protein. Aim 3 will characterize recently identified novel small molecules that bind the ETS domain of EWS-fusion proteins and inhibit its function. Medicinal chemistry approaches will be used to further refine these molecules and ultimately, in collaboration with Core B, convert these molecules into targeted protein degraders. Preclinical studies will then be performed with Core C prior to more extended development and potential clinical translation. This project will provide both biological insight into EWS-fusion protein biology and novel small molecules that directly inactivate EWS-fusion proteins. Successful completion of this project could significantly influence how we treat patients with Ewing Sarcoma. !